טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentHertz Rivka
SubjectThe Expression of Acetylchlinesterase Following Stress in
the Vertebrate Retina
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Ido Perlman
Full Thesis textFull thesis text - English Version


Abstract

Acetylcholinesterase (AChE), plays a crucial role in cholinergic synapses by hydrolyzing acetylcholine (ACh), thus terminating synaptic transmission. In addition, AChE participates in neuronal stress responses. In the retina, AChE was surprisingly demonstrated in human adult photoreceptors that are not involved in cholinergic synaptic activity, raising the possibility that it exerts stress-related morphogenic function(s). ATF3 (activating transcription factor 3) gene encodes a member of the ATF/CREB (cAMP-response-element-binding protein) family of transcription factors. ATF3 gene is an immediate early gene induced by a variety of stress signals in different cell types. ATF3 is a repressor of the cyclic adenosine mono-phosphate-responsive element (CRE)-dependent transcription.

The goal of this study was to expand on the involvement of AChE in stress response of the retina and to elucidate the signal transduction pathway involved and the interactions between AChE and ATF3.

Adult albino and pigmented mice were kept for two weeks in complete darkness to augment their susceptibility to photic stress. The mice were exposed to bright, damaging light for different periods of time: 0, 2, 10 and 24 hr. The electroretinogram (ERG) was recorded to assess retinal function. AChE mRNA expression was monitored by Real-Time PCR. Immunohistochemistry was performed to check ATF3 protein expression, and TUNEL was used to determine apoptosis. Reporter assay analysis was used for testing AChE-promoter activity.

The retina of pigmented mice showed increased expression of AChE mRNA following exposure to bright light, but only negligible signs of light-induced damage as evident from the ERG recording and TUNEL immunohistochemistry. In contrast, the retina of albino mice showed reduction of ERG responses depending upon the period of exposure. Augmented expression of AChE mRNA was found after 2 and 10 hours of exposure to bright light compared to control mice kept in darkness and not exposed to bright light. The light-exposed albino mice exhibited up-regulation of ATF3 in the inner nuclear layer (INL), but not in the distal retina. Apoptosis was observed only in the outer nuclear layer (ONL) of light exposed mice. A reporter assay analysis showing that ATF3 repressed AChE promoter activity in PC12 transfected cells.

The findings indicate that light stress induces AChE mRNA expression followed by apoptosis. The inhibitory effect of ATF3 on AChE-promoter activity in PC12 cells, could explain our in vivo observations in which apoptosis was restricted to the outer retina, while the inner retina was probably protected by ATF3 from AChE-induced apoptosis.